Molecular assay kits

ABSTRACT

Methods, kits, and compositions for evaluating the quality of nucleic acids within a biological sample for analysis in a molecular assay are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/356,368, filed May 5, 2014, which claims the benefit of InternationalApplication No. PCT/US2012/063402, filed Nov. 2, 2012, now pending,which claims the benefit of U.S. Provisional Application No. 61/556,005,filed Nov. 4, 2011, the contents of each of which applications is herebyincorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to control reagents for evaluatingbiological samples.

BACKGROUND

Access to and use of human specimens is an essential part of the cancerresearch and drug discovery infrastructure, enabling researchers to,inter alia, correlate gene mutations/polymorphisms with particularcancers, identify drug targets, develop therapeutic compounds, guidetherapy decisions, and understand drug metabolism. Research using humanspecimens can help predict drug response and toxicity, and clinicaloutcome. Many different types of biological specimens are required tosupport these studies: normal and malignant tissues, blood, bloodproducts, other bodily fluids, as well as proteins and nucleic acidsthat can be extracted from them.

Formalin-fixed, paraffin-embedded (FFPE) tissue, which is one commonmethod of preserving specimens, is a frequent source of biologicalspecimens. Formaldehyde fixation, however, causes cross-linking betweennucleic acids and proteins, the reversal of which leads to fragmentationof DNA and RNA. And, the paraffin-embedded tissue sections requirede-waxing to allow penetration by aqueous solutions prior to analysis ofnucleic acids.

For example, in one extraction procedure a razor blade is used to scrapeFFPE sections, which are then transferred into microfuge tubes forprocessing. The traditional method of paraffin removal involves organicextractions using xylene and graded alcohols. This procedure istime-consuming, cumbersome, and requires special handling, as xylene isa highly toxic chemical that emits noxious fumes.

Phase extraction de-waxing protocols are time consuming and laborious.And, the repeated handling, aspirations and tube transfers can result innon-quantitative harvests of the nucleic acids. Moreover, repeatedvortexing of the sample and exposure to harsh solvents can causeadditional sample degradation. Commercial kits are available thatoptimize the nucleic acid extraction process but the resulting qualityand quantity of nucleic acid recovered from FFPE tissues is variable.

Problems exist in the quantitation of nucleic acids from preservedclinical specimens such as FFPE tissues. Extracted nucleic acid qualityand quantity is often affected by both sample collection and extractionprocedures due to degradation and fragmentation. This can compromise,for example, the ability to measure the extracted nucleic acids.Qualitative and quantitative assay errors often result when theseextracts are evaluated by standard analytical techniques. Moreover,incomplete extractions can introduce error into calculations, such asmRNA copy number determinations.

A variety of methods exist for attempting to assess the quantity andquality of extracted nucleic acids. For example, 260 nm/280 nmabsorbance by spectrophotometry can assess high molecular weight(intact) genomic DNA. But nucleic acid fragmentation can result inhighly erroneous results and an overestimation of nucleic acid amounts.The use of intercalating dyes is also widely used, however, the accuracyof assays based on these dyes are significantly impacted by anover-abundance of small nucleic acid fragments in degraded sample.

Sensitivity of detection of a genetic variant is dependent upon thequantity and quality of nucleic acid available for analysis. Althoughexisting methods that are used to determine nucleic acid quantity andquality are useful in limited circumstances, they generally involveadditional testing prior to genetic analysis of a sample. A simplemethod to evaluate the quantity and quality of DNA or RNA simultaneouslywith the genetic analysis is therefore needed.

SUMMARY OF THE INVENTION

A kit is provided for evaluating a biological sample containingpotentially degraded DNA, comprising a control reagent and a targetamplification reagent, wherein the control amplification reagentcomprises one or more pairs of amplification oligonucleotides capable ofamplifying a medium chain acyl-coenzyme A dehydrogenase (MCAD) nucleicacid molecule or a complement thereof, and wherein the targetamplification reagent comprises one or more pairs of amplificationoligonucleotides capable of amplifying a non-MCAD target gene ofinterest or a complement thereof. Frequently, one or more of theamplification oligonucleotides is/are labeled. Also frequently, thebiological sample comprises a tissue sample, which often comprises aformalin-fixed paraffin-embedded (FFPE) tissue sample.

In certain embodiments two or more pairs of amplificationoligonucleotides are provided that are capable of amplifying an MCADnucleic acid molecule or a complement thereof. In frequent embodimentsthree or more pairs of amplification oligonucleotides are provided thatare capable of amplifying an MCAD nucleic acid molecule or a complementthereof. In frequent embodiments each of the two or more pairs ofamplification oligonucleotides comprises a sense amplificationoligonucleotide and an antisense oligonucleotide, and wherein the senseamplification oligonucleotide of each of the two or more pairs ofamplification oligonucleotides comprises the same oligonucleotidesequence. Also frequently, each of the two or more pairs ofamplification oligonucleotides comprises a sense amplificationoligonucleotide and an antisense oligonucleotide, and wherein theantisense amplification oligonucleotide of each of the two or more pairsof amplification oligonucleotides comprises the same oligonucleotidesequence. Often, each pair of amplification oligonucleotides is capableof amplifying a region of the MCAD nucleic acid molecule to produce anMCAD amplicon, and wherein each MCAD amplicon produced by each pair ofamplification oligonucleotides is detectably distinguishable from theMCAD amplicon produced by each other pair of amplificationoligonucleotides.

In frequent embodiments each MCAD amplicon produced by each pair ofamplification oligonucleotides is detectably distinguishable from theMCAD amplicon produced by each other pair of amplificationoligonucleotides on the length of each MCAD amplicon.

Methods for evaluating the quality of a biological sample are alsoprovided, comprising: (a) contacting a nucleic acid molecule obtainedfrom a tissue sample with a control amplification reagent to form areaction mixture, wherein the nucleic acid molecule comprises a highlyconserved region of a control nucleic acid; (b) subjecting the reactionmixture to amplification conditions to produce an amplification mixture,whereby two or more portions of the highly conserved region of thecontrol nucleic acid are amplified to produce two or more detectablydistinguishable control amplicons, each having a different length,wherein the two or more different length control amplicons form a sizecontrol ladder comprised of amplicons of increasing length; and (c)evaluating the amplification mixture to detect the length and amount ofeach control amplicon, wherein the biological sample is determined tocontain degraded nucleic acid molecules that may adversely impactmolecular analysis of the biological sample if: (1) the amount detectedof each the two or more control amplicons is smaller than the amountdetected of each the two or more control amplicons in a comparativecontrol sample; (2) the amount detected of each of the two or morecontrol amplicons in the size control ladder decreases as the length ofeach control amplicon in the size control ladder increases; or (3) oneor more of the control amplicons are undectable after production of theamplification mixture. Frequently, the biological sample comprises atissue sample such as an FFPE tissue sample.

In certain frequent embodiments the control nucleic acid comprises MCAD.

Frequently, the results of the method are utilized to evaluate amolecular assay involving the biological sample. Also frequently theresults of the method are utilized to reject the results of a molecularassay involving the biological sample.

Methods of conducting an amplification assay are also provided,comprising: (a) contacting a biological sample with a controlamplification reagent and a target amplification reagent to form areaction mixture, wherein the control amplification reagent is capableof taking part in a nucleic acid amplification reaction involving ahighly conserved region of a control nucleic acid or a portion thereof,if present in the biological sample, and wherein the targetamplification reagent is capable of taking part in a nucleic acidamplification reaction involving a target nucleic acid or a portionthereof, if present in the biological sample; (b) subjecting thereaction mixture to amplification conditions to produce an amplificationmixture, whereby a portion of the highly conserved region of the controlnucleic acid and the target nucleic acid or portion thereof, if present,are amplified to produce a detectable control amplicon and a detectabletarget amplicon; (c) evaluating the amplification mixture to detect thecontrol amplicon and the target amplicon; and (d) comparing the detectedcontrol amplicon with the detected target amplicon to evaluate theamount and quality of the target nucleic acid or portion thereof and/orthe highly conserved region of the control nucleic acid or portionthereof, present in the biological sample. In certain embodiments thetarget nucleic acid and/or control nucleic acid comprises genomic DNA.

In certain frequent embodiments the control nucleic acid comprises MCAD.

Also frequently, the control amplification reagent comprisesamplification oligonucleotides for use in producing two or moredetectably distinguishable control amplicons, each having a differentlength, wherein the two or more different length control amplicons forma size control ladder comprised of amplicons of increasing length.Often, the target amplification reagent comprises amplificationoligonucleotides for use in producing one or more detectable ampliconsfor each target nucleic acid.

The target nucleic acid reagent of the present kits and methods is notlimited. However, often the target nucleic acid reagent comprises areagent specific for wild-type or mutant v-Raf murine sarcoma viraloncogene homolog B1 (BRAF), V-Ki-ras2 Kirsten rat sarcoma viral oncogenehomolog (KRAS), epidermal growth factor receptor (EGFR),phosphatidylinositol 3-kinase (PIK3CA), phosphatase and tensin homolog(PTEN), v-akt murine thymoma viral oncogene homolog (AKT), anaplasticlymphoma kinase (ALK), mast/stem cell growth factor receptor (c-Kit),neuroblastoma RAS viral oncogene homolog (NRAS), met proto-oncogenehepatocyte growth factor receptor (c-Met), prostate cancer gene 3(PCA3), prostate specific membrane antigen (PSMA), prostate specificantigen (PSA), tumor protein 53 (TP53), Echinodermmicrotubule-associated protein-like 4 (EML4), EML4-ALK fusions, androgenregulated gene-ETS family member gene fusions, RAF gene fusions,breakpoint cluster region −V-abl Abelson murine leukemia viral oncogenehomolog 1 fusions (BCR-Abl), cytochrome P450 2D6 (CYP2D6), cytochromeP450 2C19 (CYP2C19), cytochrome P450 2C9 (CYP2C9), vitamin K epoxidereductase complex subunit 1 (VKORC1), thiopurine methyltransferase(TMPT), bilirubin UDP-glucuronosyltransferase isozyme 1 (UGT1A1), and/orATP-binding cassette sub-family B member 1 (ABCB1), or combinationsthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, FIG. 1B, FIG. 1C depict exemplary results of an assay of goodquality (FIG. 1A), acceptable quality (FIG. 1B), and very degraded DNA(FIG. 1C) using control reagents of the present disclosure. TheX-coordinate depicts amplicon size, and the Y-coordinate depictsfluorescence intensity in RFUs.

FIG. 2A, FIG. 2B, FIG. 2C depict exemplary results of an assay ofsufficient DNA (FIG. 2A), reduced DNA (FIG. 2B), and very little DNA(FIG. 2C) in a sample using control reagents of the present disclosure.The X-coordinate depicts amplicon size, and the Y-coordinate depictsfluorescence intensity in RFUs.

FIG. 3A, FIG. 3B, FIG. 3C depict exemplary results of an assay formutant DNA and control DNA. The X-coordinate depicts amplicon size, andthe Y-coordinate depicts fluorescence intensity in RFUs.

FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D depict results of an assay of controlbalancing with 100% wildtype DNA. The X-coordinate depicts ampliconsize, and the Y-coordinate depicts fluorescence intensity in RFUs.

FIG. 5A, FIG. 5B, FIG. 5C depict results of an assay of controlbalancing with 20% mutant DNA in wildtype DNA. The X-coordinate depictsamplicon size, and the Y-coordinate depicts fluorescence intensity inRFUs.

FIG. 6 depicts exemplary results of an assay of good quality andsufficient amounts of DNA using control reagents of the presentdisclosure. The X-coordinate depicts amplicon size, and the Y-coordinatedepicts fluorescence intensity in RFUs.

FIG. 7 depicts exemplary results of an assay of degraded and loweramounts of DNA using control reagents of the present disclosure. TheX-coordinate depicts amplicon size, and the Y-coordinate depictsfluorescence intensity in RFUs.

FIG. 8 depicts exemplary results of an assay identifying multiple mutantDNA amplicons in addition to control DNA. The X-coordinate depictsamplicon size, and the Y-coordinate depicts fluorescence intensity inRFUs.

FIG. 9A and FIG. 9B depict the mRNA sequence of Medium Chain Acyl-CoADehydrogenase gene or “MCAD,” GenBank Accession Number NM_001127328.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all terms of art, notations and otherscientific terms or terminology used herein have the same meaning as iscommonly understood by one of ordinary skill in the art to which thisdisclosure belongs. Many of the techniques and procedures described orreferenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted. All patents,applications, published applications and other publications referred toherein are incorporated by reference in their entirety. If a definitionset forth in this section is contrary to or otherwise inconsistent witha definition set forth in the patents, applications, publishedapplications, and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

The term “about” is used herein to mean approximately, roughly, around,or in the region of. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the values set forth. In general, the term “about” isused herein to modify a numerical value above and below the stated valueby a deviation of 20 percent.

As used herein, the word “or” means any one member of a particular listand also includes any combination of members of that list.

As used herein, the terms “detect,” “detecting” or “detection” maydescribe either the general act of discovering or discerning or thespecific observation of a composition, whether it is labeled orotherwise. In frequent embodiments, the detection process involvesevaluating something (e.g., a sample, a reaction product, etc.) for thepresence of a detectable signal (e.g., fluorescence) attributable to thepresence of a particular analyte. A particular detectable signal levelor strength (e.g., strong or large relative fluorescence levels) oftendenotes a particular predetermined characteristic of the analyte such asanalyte amount, analyte size, sample quality, etc.

“Nucleic acid” or “nucleic acid molecule” refers to a multimericcompound comprising nucleotides or analogs which have nitrogenousheterocyclic bases or base analogs linked together to form apolynucleotide, including conventional RNA, DNA, mixed RNA-DNA, andpolymers that are analogs thereof. A nucleic acid “backbone” may be madeup of a variety of linkages, including one or more ofsugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptidenucleic acids” or PNA; PCT No. WO 95/32305), phosphorothioate linkages,methylphosphonate linkages, or combinations thereof. Sugar moieties of anucleic acid may be ribose, deoxyribose, or similar compounds withsubstitutions, e.g., 2′ methoxy or 2′ halide substitutions. Nitrogenousbases may be conventional bases (A, G, C, T, U), analogs thereof (e.g.,inosine or others; see THE BIOCHEMISTRY OF THE NUCLEIC ACIDS 5-36 (Adamset al., ed., 11^(th) ed., 1992), derivatives of purines or pyrimidines(e.g., N⁴-methyl deoxygaunosine, deaza- or aza-purines, deaza- oraza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6position, purine bases with a substituent at the 2, 6 or 8 positions,2-amino-6-methylaminopurine, O.sup.6-methylguanine, 4-thio-pyrimidines,4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, andO.sup.4-alkyl-pyrimidines; U.S. Pat. No. 5,378,825 and PCT No. WO93/13121). Nucleic acids may include one or more “abasic” residues wherethe backbone includes no nitrogenous base for one or more positions(U.S. Pat. No. 5,585,481). A nucleic acid may include only conventionalRNA or DNA sugars, bases and linkages, or may include both conventionalcomponents and substitutions (e.g., conventional bases with 2′ methoxylinkages, or polymers containing both conventional bases and analogs).The term includes “locked nucleic acid” (LNA), an analogue containingone or more LNA nucleotide monomers with a bicyclic furanose unit lockedin an RNA mimicking sugar conformation, which enhance hybridizationaffinity for complementary RNA and DNA sequences (Vester et al.,Biochemistry, 2004, 43(42):13233-41). Embodiments of oligomers that mayaffect stability of a hybridization complex include PNA oligomers,oligomers that include 2′-methoxy or 2′-fluoro substituted RNA, oroligomers that affect the overall charge, charge density, or stericassociations of a hybridization complex, including oligomers thatcontain charged linkages (e.g., phosphorothioates) or neutral groups(e.g., methylphosphonates).

“Oligomer” or “oligonucleotide” refers to a nucleic acid of generallyless than 1,000 nucleotides (nt), including those in a size range havinga lower limit of about 2 to 5 nt and an upper limit of about 500 to 900nt. Some preferred embodiments are oligomers in a size range with alower limit of about 5 to 15 nt and an upper limit of about 50 to 600nt, and other preferred embodiments are in a size range with a lowerlimit of about 10 to 20 nt and an upper limit of about 22 to 100 nt.Oligomers may be purified from naturally occurring sources, butpreferably are synthesized by using any well-known enzymatic or chemicalmethod. Oligomers may be referred to by functional names (e.g., captureprobe, primer or promoter primer) which are understood to refer tooligomers.

The term “gene” refers to a nucleic acid molecule that comprisesnon-coding and coding sequences, or just coding sequences necessary forthe production of a polypeptide, precursor, or RNA. The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence. The term also encompasses the coding region of astructural gene and the sequences located adjacent to the coding regionon both the 5′ and 3′ ends for a distance of about 1 kb or more oneither end such that the gene corresponds to the length of thefull-length mRNA. Sequences located 5′ of the coding region and presenton the mRNA are referred to as 5′ non-translated sequences. Sequenceslocated 3′ or downstream of the coding region and present on the mRNAare referred to as 3′ non-translated sequences. The term “gene”encompasses both cDNA and genomic forms of a gene. A genomic form orclone of a gene contains the coding region interrupted with non-codingsequences termed “introns” or “intervening regions” or “interveningsequences.”

A “sample” or “specimen” refers to any composition in which a control ortarget nucleic acid may exist as part of a mixture of components, e.g.,in water or environmental samples, food stuffs, materials collected forforensic analysis, or biopsy samples for diagnostic testing. “Biologicalsample” refers to any tissue or material derived from a living or deadorganism which may contain a target nucleic acid, including, e.g.,cells, tissues, lysates made from cells or tissues, sputum, peripheralblood, plasma, serum, cervical swab samples, biopsy tissues (e.g., lymphnodes), respiratory tissue or exudates, gastrointestinal tissue, urine,feces, semen, or other fluids or materials. A sample may be treated tophysically disrupt tissue and/or cell structure to release intracellularcomponents into a solution which may contain enzymes, buffers, salts,detergents and other compounds, such as are used to prepare a sample foranalysis by using standard methods. The sample may require preliminaryprocessing designed to purify, isolate, and/or enrich the sample for thetarget(s) or cells that contain the target(s). A variety of techniquesknown to those of ordinary skill in the art may be used for thispurpose, including but not limited to: centrifugation; immunocapture;cell lysis; and, nucleic acid target capture (See, e.g., EP Pat. No.1409727, incorporated herein by reference).

As used herein, the “Medium Chain Acyl-CoA Dehydrogenase” gene or “MCAD”refers to the gene referenced in GenBank Accession Number NM_001127328.The MCAD gene maps to chromosome 1p31, comprises 12 exons spanning 44 kbof DNA, and encodes a 2627 base mRNA transcript. See FIG. 9A & FIG. 9B;Matsubara et al., Proc Nat'l Acad. Sci. USA, 1986, Vol. 83, No. 17, pp.6543-47, incorporated herein by reference. MCAD is an exemplary “controlnucleic acid.”

While MCAD control nucleic acids are specifically exemplified in thepresent disclosure, the invention is not limited to the use of this geneas a control nucleic acid. In particular, genes having regions withminimal sequence variability or highly conserved regions and that arepresent in a subject despite the co-presence of one or more mutations orpolymorphisms in certain target genes, a cancerous or pre-cancerouscondition, or a foreign pathogenic gene, may provide useful controlnucleic acids according to the present methods. In such circumstances itis particularly useful to target regions of one or more of these othercontrol genes that are highly conserved, having minimal or no knownsequence variability, for amplification. “Control amplificationreagents” are utilized in nucleic acid amplification reactions involvingcontrol nucleic acids of the present disclosure to amplify the controlnucleic acid or highly conserved region thereof. In this process, underamplification conditions, particular portions of the control nucleicacid are targeted with one or more amplification oligonucleotides andsubject to amplification reactions, thereby amplifying (i.e., increasingcopy number) the targeted portion of the control nucleic acid.

“Nucleic acid amplification,” “amplification reaction,” or“amplification” refers to any known procedure for obtaining multiplecopies of a target nucleic acid sequence or its complement or fragmentsthereof. The multiple copies may be referred to as amplicons oramplification products. Amplification of “fragments” refers toproduction of an amplified nucleic acid that contains less than thecomplete target nucleic acid or its complement, e.g., produced by usingan amplification oligonucleotide that hybridizes to, and initiatespolymerization from, an internal position of the target nucleic acid.Known amplification methods include, for example, replicase-mediatedamplification, polymerase chain reaction (PCR), ligase chain reaction(LCR), strand-displacement amplification (SDA), andtranscription-mediated or transcription-associated amplification.Replicase-mediated amplification uses self-replicating RNA molecules,and a replicase such as QB-replicase (e.g., U.S. Pat. No. 4,786,600).PCR amplification uses a DNA polymerase, pairs of primers, and thermalcycling to synthesize multiple copies of two complementary strands ofdsDNA or from a cDNA (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and4,800,159). LCR amplification uses four or more differentoligonucleotides to amplify a target and its complementary strand byusing multiple cycles of hybridization, ligation, and denaturation(e.g., U.S. Pat. Nos. 5,427,930 and 5,516,663). SDA uses a primer thatcontains a recognition site for a restriction endonuclease and anendonuclease that nicks one strand of a hemimodified DNA duplex thatincludes the target sequence, whereby amplification occurs in a seriesof primer extension and strand displacement steps (e.g., U.S. Pat. Nos.5,422,252; 5,547,861; and 5,648,211).

“Hybridization conditions” refer to the cumulative physical and chemicalconditions under which nucleic acid sequences that are completely orpartially complementary form a hybridization duplex or complex, usuallyby standard base pairing. Such conditions are well known to thoseskilled in the art, are predictable based on sequence composition of thenucleic acids involved in hybridization complex formation, or may bedetermined empirically by using routine testing (e.g., Sambrook et al.,MOLECULAR CLONING, A LABORATORY MANUAL (2^(nd) ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) at §§ 1.90-1.91,7.37-7.57, 9.47-9.51, and 11.47-11.57, particularly §§ 9.50-9.51,11.12-11.13, 11.45-11.47 and 11.55-11.57 (incorporated herein byreference)).

“Sufficiently complementary” means that a contiguous nucleic acid basesequence is capable of hybridizing to another base sequence by standardbase pairing (hydrogen bonding) between a series of complementary bases.Complementary sequences may be completely complementary at each positionin an oligomer sequence relative to its target sequence by usingstandard base pairing (e.g., G:C, A:T or A:U pairing) or sequences maycontain one or more positions that are not complementary by base pairing(including abasic residues), but such sequences are sufficientlycomplementary because the entire oligomer sequence is capable ofspecifically hybridizing with its target sequence in appropriatehybridization conditions. Contiguous bases in an oligomer are at least80%, preferably at least 90%, and more preferably completelycomplementary to the intended target sequence.

“Label” refers to a moiety or compound that is detected or leads to adetectable signal. Any detectable moiety may be a label, e.g.,radionuclide, ligand such as biotin or avidin, enzyme, enzyme substrate,reactive group, chromophore such as a dye or particle (e.g., latex ormetal bead) that imparts a detectable color, luminescent compound (e.g.bioluminescent, phosphorescent or chemiluminescent compound), andfluorescent compound.

A “target nucleic acid” as used herein is a nucleic acid comprising a“target sequence” to be amplified. Target nucleic acids may be DNA orRNA as described herein, and may be either single-stranded ordouble-stranded. The target nucleic acid may include other sequencesbesides the target sequence, which may not be amplified. Typical targetnucleic acids include virus genomes, bacterial genomes, fungal genomes,plant genomes, animal genomes, rRNA, tRNA, or mRNA from viruses,bacteria or eukaryotic cells, mitochondrial DNA, or chromosomal DNA.“Target amplification reagents” are utilized in nucleic acidamplification reactions involving target nucleic acids of the presentdisclosure to amplify the target nucleic acid or portion thereof. Inthis process, under amplification conditions, one or more particularportions of the target nucleic acid is/are targeted with one or moreamplification oligonucleotides and subject to amplification reactions,thereby amplifying (i.e., increasing copy number) the targeted portionof the target nucleic acid.

The term “target sequence” as used herein refers to the particularnucleotide sequence of the target nucleic acid that is to be amplifiedand/or detected. The “target sequence” includes the complexing sequencesto which oligonucleotides (e.g., priming oligonucleotides and/orpromoter oligonucleotides) complex during the processes ofamplification. Where the target nucleic acid is originallysingle-stranded, the term “target sequence” will also refer to thesequence complementary to the “target sequence” as present in the targetnucleic acid. Where the target nucleic acid is originallydouble-stranded, the term “target sequence” refers to both the sense (+)and antisense (−) strands. In choosing a target sequence, the skilledartisan will understand that a “unique” sequence should be chosen so asto distinguish between unrelated or closely related target nucleicacids.

“Target binding sequence” is used herein to refer to the portion of anoligomer that is configured to hybridize with a target nucleic acidsequence. Preferably, the target binding sequences are configured tospecifically hybridize with a target nucleic acid sequence. Targetbinding sequences may be 100% complementary to the portion of the targetsequence to which they are configured to hybridize; but not necessarily.Target-binding sequences may also include inserted, deleted and/orsubstituted nucleotide residues relative to a target sequence. Less than100% complementarity of a target binding sequence to a target sequencemay arise, for example, when the target nucleic acid is a plurality ofstrains within a species. It is understood that other reasons exist forconfiguring a target binding sequence to have less than 100%complementarity to a target nucleic acid.

The term “configured to” denotes an actual arrangement of thepolynucleotide sequence configuration of the references oligonucleotidetarget hybridizing sequence. For example, amplification oligomers thatare configured to generate a specified amplicon from a target sequencehave polynucleotide sequences that hybridize to the target sequence andcan be used in an amplification reaction to generate the amplicon. Alsoas an example, oligonucleotides that are configured to specificallyhybridize to a target sequence have a polynucleotide sequence thatspecifically hybridizes to the referenced sequence under stringenthybridization conditions.

The phrase “configured to specifically hybridize to” as used hereinmeans that the target hybridizing region of an amplificationoligonucleotide, detection probe or other oligonucleotide is designed tohave a polynucleotide sequence that could target a sequence of the atarget nucleic acid or control nucleic acid, or specific portionsthereof. Such an oligonucleotide is not limited to targeting thatsequence only, but is rather useful as a composition, in a kit or in amethod for the control and target nucleic acids. The oligonucleotide isdesigned to function as a component of an assay for amplification anddetection of the control nucleic acid or target nucleic acid in asample, and therefore is designed to target a specific control nucleicacid or target nucleic acid in the presence of other nucleic acidscommonly found in testing samples. “Specifically hybridize to” does notmean exclusively hybridize to, as some small level of hybridization tonon-target nucleic acids may occur, as is understood in the art. Rather,“specifically hybridize to” means that the oligonucleotide is configuredto function in an assay to primarily hybridize the target so that anaccurate detection of target nucleic acid in a sample can be determined.

An “amplification oligomer” or “amplification oligonucleotide” is anoligomer, at least the 3′-end of which is complementary to a targetnucleic acid, and which hybridizes to a target nucleic acid, or itscomplement, and participates in a nucleic acid amplification reaction.An example of an amplification oligomer is a “primer” that hybridizes toa target nucleic acid and contains a 3′ OH end that is extended by apolymerase in an amplification process. Another example of anamplification oligomer is an oligomer that is not extended by apolymerase (e.g., because it has a 3′ blocked end) but participates inor facilitates amplification. For example, the 5′ region of anamplification oligonucleotide may include a promoter sequence that isnon-complementary to the target nucleic acid (which may be referred toas a “promoter-primer” or “promoter provider”). Those skilled in the artwill understand that an amplification oligomer that functions as aprimer may be modified to include a 5′ promoter sequence, and thusfunction as a promoter-primer. Incorporating a 3′ blocked end furthermodifies the promoter-primer, which is now capable of hybridizing to atarget nucleic acid and providing an upstream promoter sequence thatserves to initiate transcription, but does not provide a primer foroligo extension. Such a modified oligo is referred to herein as a“promoter provider” oligomer. Size ranges for amplificationoligonucleotides include those that are about 10 to about 70 nt long(not including any promoter sequence or poly-A tails) and contain atleast about 10 contiguous bases, or even at least 12 contiguous basesthat are complementary to a region of the target nucleic acid sequence(or a complementary strand thereof). The contiguous bases are at least80%, or at least 90%, or completely complementary to the target sequenceto which the amplification oligomer binds. An amplification oligomer mayoptionally include modified nucleotides or analogs, or additionalnucleotides that participate in an amplification reaction but are notcomplementary to or contained in the target nucleic acid, or templatesequence. It is understood that when referring to ranges for the lengthof an oligonucleotide, amplicon or other nucleic acid, that the range isinclusive of all whole numbers (e.g. 19-25 contiguous nucleotides inlength includes 19, 20, 21, 22, 23, 24 & 25).

The term “amplicon” or the term “amplification product” as used hereinrefers to the nucleic acid molecule generated during an amplificationprocedure that is complementary or homologous to a sequence containedwithin the target sequence. The complementary or homologous sequence ofan amplicon is sometimes referred to herein as a “target-specificsequence.” Amplicons generated using the amplification oligomers of thecurrent invention may comprise non-target specific sequences. Ampliconscan be double stranded or single stranded and can include DNA, RNA orboth. For example, DNA-dependent RNA polymerase transcribes singlestranded amplicons from double stranded DNA duringtranscription-mediated amplification procedures. These single strandedamplicons are RNA amplicons and can be either strand of a doublestranded complex; depending on how the amplification oligomers areconfigured. RNA-dependent DNA polymerases synthesize a DNA strand thatis complementary to an RNA template. Thus, amplicons can be doublestranded DNA and RNA hybrids. RNA-dependent DNA polymerases ofteninclude RNase activity, or are used in conjunction with an RNase, whichdegrades the RNA strand. Thus, amplicons can be single stranded DNA.RNA-dependent DNA polymerases and DNA-dependent DNA polymerasessynthesize complementary DNA strands from DNA templates. Thus, ampliconscan be double stranded DNA. RNA-dependent RNA polymerases synthesize RNAfrom an RNA template. Thus, amplicons can be double stranded RNA. DNADependent RNA polymerases synthesize RNA from double stranded DNAtemplates, also referred to as transcription. Thus, amplicons can besingle stranded RNA. Amplicons and methods for generating amplicons areknown to those skilled in the art. For convenience herein, a singlestrand of RNA or a single strand of DNA may represent an amplicongenerated by an amplification oligomer combination of the currentinvention. Such representation is not meant to limit the amplicon to therepresentation shown. Skilled artisans in possession of the instantdisclosure will use amplification oligomers and polymerase enzymes togenerate any of the numerous types of amplicons; all within the spiritof the current invention.

As used herein “highly conserved” or “highly conserved regions” refersto nucleic acid sequences that vary in less than 1/100 or 1/1000 membersof the population.

Tissue Samples

According to the present disclosure nucleic acids can be isolated fromany biological sample using conventional methods. Thus, tissue samplesof any origin may be utilized according to the present methods. Duringpreservation, tissue samples are often treated with a fixative agentthat causes protein cross-linking. Fixatives such as aldehyde fixatives(e.g., formalin/formaldehyde, glutaraldehyde) are typically used; butother fixatives are contemplated within the scope of the disclosure,such as alcohol immersion (see, e.g., Battifora & Kopinski, J.Histochem. Cytochem., 1986, 34:1095, incorporated herein by reference),oxidizing agents (e.g., osmium tetroxide, potassium dichromate, chromicacid, and potassium permanganate), mercurials (e.g., B-5 and Zenker's),picrates, and HOPE fixative (Hepes-glutamic acid buffer-mediated organicsolvent protection effect). The main action of aldehyde fixatives is tocross-link amino groups in proteins through the formation of CH₂(methylene) linkage (when using formaldehyde) or a C₅H₁₀ cross-link(when using glutaraldehyde). This process, while preserving thestructural integrity of the cells and tissue, degrades RNA and DNApresent in the tissue.

Tissue samples are also frequently fixed and then embedded in paraffin(i.e., wax). According to the present invention, nucleic acids can beisolated from any wax-embedded biological tissue sample by conventionalmethods. In one embodiment, the samples are both formalin-fixed andparaffin-embedded.

Microtomy is frequently used to slice FFPE samples into fine sections.DNA is extracted from the sliced sections using, for example,commercially-available FFPE DNA extraction kits (e.g., QIAMP FFPE DNAExtraction Kit, Qiagen GmbH, Hilden, Germany). DNA from these samples istypically degraded due to the action of the formalin fixative. Moreover,the amount of DNA in the sliced sample often varies greatly and anyparticular section of tissue often contains a mixture of DNA types, forexample a mixture of mutant DNA and wildtype DNA. The present methodsand compositions are sensitive enough to permit the detection of atleast about 1% mutant DNA in an otherwise wild-type sample.

In an exemplary extraction procedure paraffin is dissolved in xylene andremoved. The sample is then lysed under denaturing conditions with ashort proteinase K digestion and incubated at 90° C. to reverse formalincross-linking. DNA binds to the membrane and contaminants flow throughor are washed away. Purified and concentrated DNA is then eluted inbuffer or water from the membrane.

In a frequent embodiment the purified and concentrated DNA comprisesMedium Chain Acyl-CoA Dehydrogenase (MCAD) gene DNA in addition to oneor more target nucleic acid molecules.

Exemplary Assays

FIG. 1A describes the results of an exemplary post-amplification gelelectrophoresis assay of an amplified and purified targetoligonucleotide in addition to three different MCAD oligonucleotides of100 bases, 150 bases, and 200 bases. In this example, the MCAD ampliconswere prepared utilizing three different unlabeled reverse primers and asingle labeled forward primer. This figure depicts equal signalintensities of each of the MCAD controls, here at about 3000 RFUs. Thetarget peak (e.g., from a KRAS mutant) can be visualized having a lowerintensity and lying between the 100 base and 150 base peaks of the MCADcontrols.

Although control amplicons of 100, 150, and 200 bases are specificallyexemplified, the particular sizes of the control amplicons are notlimited. In particular, any particular selection of amplicon sizes maybe chosen provided that at least two different control amplicons arederived from the same gene. Preferably at least three control ampliconsare derived from the same gene, provided they have different sizes thatare discernable from one another by, for example, gel electrophoresis.In frequent embodiments, the sizes of the control amplicons are chosenwith reference to the one or more target genes that are to be assayed toensure that the target gene amplicon(s) are not co-extensive insize/length with one or more of the control peaks. When mutations orpolymorphisms are targeted by the assay, often the sizes of the controlamplicons are chosen depending on the number and sizes of targetamplicons, each containing one or more polymorphisms, which could bepresent in any particular assay. For example, a particular sample mayharbor one or more polymorphisms targeted by a multi-marker assay and,in order to minimize the number of target primers utilized, or to designthe most appropriate primers, choices must be made about various targetamplicon sizes. Often in such circumstances it is advantageous to choosethe control amplicon sizes/lengths in a manner that does not interferewith target primer design choices. Thus, control amplicon sizes/lengthscan vary within any size range that is practical to the assay beingperformed.

In frequently preferred embodiments the control reagents, such as MCADcontrols, are provided in a multiplex format such that multipledifferent-sized amplicons are produced and detected from the same targetcontrol gene to provide a control ladder.

The control peaks provide a reference against which the analytical peakscan be compared allowing the proportion of the amplicon of interest tobe measured (see, e.g., FIGS. 1A, 1B, 1C, 2A, 2B, 2C, 3A, 3B, 3C, 5A,5B, 5C, 8). This is important when dealing with mixed cell populations.In addition, the control peaks provide a measure of the amplifiabilityand concentration of the sample DNA (see FIGS. 1A, 1B, 1C, 2A, 2B, 2C,3A, 3B, 3C). For example, the controls of the present invention, sincethey are internal controls originating from the same sample beingassayed for a particular target gene, signal the degredation state ofthe overall sample. These aspects are important when dealing withinhibited and/or degraded samples to achieve maximum sensitivity of theassay. In this regard, FIG. 1A depicts a post-amplification, gelelectrophoresis assay of good quality DNA. FIG. 1B depicts apost-amplification, gel electrophoresis assay of acceptable quality DNA.And, FIG. 1C depicts a post-amplification, gel electrophoresis assay ofvery degraded DNA, showing the loss of the third control peak (200bases) and near-loss of the second control peak (150 bases). FIG. 2A,FIG. 2B, FIG. 2C, in turn, depict the results of post-amplification, gelelectrophoresis assays of a sample having sufficient DNA levels formaximum assay sensitivity (FIG. 2A), a sample having a reduced DNAlevels that may limit assay sensitivity (FIG. 2B), and a sample havingvery little DNA that will reduce assay sensitivity.

Overall, using a multiplexed internal PCR control provides an indicationof the degree of degradation of the sample DNA. The control is typicallyused in an amplification assay, which typically presents a number ofanalysis challenges. For example, the target sequence may not be presentin every cell of a given tissue sample. In the case of an FFPE tissuesample, as indicated above, the sample has been treated in a manner thatresults in heavily degraded DNA in the sample. Similarly, preservedtissue samples may contain chemicals that act as PCR inhibitors, such asheparin, which typically interfere with PCR assays and results.

The internal controls of the present disclosure were designed to accountfor each of the above factors and to provide a variety of benefits. Forexample, by establishing a specification for the performance of thecontrol reaction, it has been possible to establish a lower level ofsensitivity for the target reaction.

Since the internal controls act as a reference value for the targetreaction, a measure of quantity and quality is also provided. In certainembodiments, the internal control peaks represent 100% of the nucleicacids in the sample being tested. In other words, the control peaks arerepresentative of the amplifiable amount of wild-type and mutant nucleicacids in the sample. In other embodiments, the internal control peaksattributable to the internal control, e.g., MCAD-type controls,represent between about 50% to about 100% of nucleic acids in the samplebeing tested. The sensitivity of detection of a target nucleic acidmolecule (e.g., a tumor-derived nucleic acid molecule) within abackground of non-target nucleic acid molecules can be determined bycomparing the target nucleic acid molecule signal (i.e., peak height) tointernal control peak heights. When fluorescent labeling schemes areutilized the peak heights are often represented by particular RFUreadings.

In a particular set of embodiments the balance of the assay is adjustedso that the internal control peak heights are equal to target sequencepeak heights in the presence of 20% of target sequence in the sample.For example, FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D depict certain examplesof balancing the control peak levels with 100% wild-type nucleic acidsin a sample. Thus, in this example, if an unknown sample is tested andthe target signal is equivalent to the internal control peak the amountof target sequence present in the sample is determined to be 20%.Similarly, if the target peak is 1/20^(th) of the signal of the controlpeak the amount of target sequence is determined to be 1%. Overall, thecontrol peak signal is independent of the target nucleic acid signal andis most frequently designed to provide a peak intensity level that isindicative of the total amount of amplifiable nucleic acids in thesample, regardless of the amount of target nucleic acid in the sample.Comparison of target signal to internal control signal can thereforeprovide useful information regarding the sensitivity of detection of theassay. To achieve a sensitivity of 1% the measurable peak is generallydiscernable above background. Though background signals may vary, oneexemplary level of background signal is about 100 RFUs and the internalcontrol signal is at least about 2000 RFUs. The internal control assay,therefore, can operate to validate the sensitivity limit with eachindividual sample; for example, as depicted in FIG. 2A, FIG. 2B, FIG.2C, FIG. 3A, FIG. 3B, FIG. 3C, FIG. 5A, FIG. 5B, FIG. 5C.

FIG. 3A depicts the formulation of the control peak to haveapproximately equal intensity with a mutant sequence peak, when themutant is present at 20%. In other words, when the target is present inthe sample at 20% of the total tested DNA, its peak height isapproximately equal to the peak height of the control. FIG. 3B shows thedetection of a 1% mutant sequence peak, which is 1/20^(th) of thecontrol peak. FIG. 3C shows a sample with a low concentration ofamplifiable nucleic acids (determined by virtue of the low controlpeaks). The situation depicted in FIG. 3C, with the low level ofamplifiable nucleic acids, would present complications in identifying a1% level of mutant DNA in the sample. With regard to FIG. 5A, FIG. 5B,FIG. 5C, these show exemplary control peaks in combination with targetnucleic acid (i.e., mutant KRAS) peaks. The mutant peaks are circled andrepresent about, or up to, 20% of the total DNA tested.

Moreover, with the presently described methods and compositions, it isalso possible to estimate the amplifiability of the sample nucleic acidsby comparing the signal strength of the control to the nominal amount ofnucleic acids present in the sample (see, e.g., FIG. 1A, FIG. 1B, FIG.1C, FIG. 2A, FIG. 2B, FIG. 2C, FIG. 3A, FIG. 3B, FIG. 3C). In thisregard, PCR amplification is often inhibited by certain chemicals,proteins, and/or contaminants that are co-purified during the nucleicacid extraction process. Such contaminating materials act on thepolymerase enzyme responsible for nucleic acid amplification. Bothtarget and internal control amplification are equally affected by thisinhibition process resulting in an equal reduction in the measurableamplified signals of internal control and target sequences. As such, useof the present internal controls eliminates the need to quantify theamplifiability of sample nucleic acids.

The presently described methods and compositions also allow aqualitative indication of the degree that nucleic acids within a samplehave degraded (see, e.g., FIG. 1A, FIG. 1B, FIG. 1C, FIG. 6, FIG. 7).For example, in certain embodiments three amplicons of differing size(for example 100, 150 and 200 bases) are amplified using reagentsspecific for the particular sized nucleic acid molecule. The efficiencyat which each sized amplicon is amplified is dependent upon the amountof nucleic acid molecule fragments of appropriate size available foramplification. DNA extracted from FFPE samples is generally subject tosignificant degradation, which results in the extracted DNA fragmentsbeing reduced in size. In general, the more highly degraded the sampleis, the smaller the fragment sizes. A comparison of the internal controlpeaks, each corresponding to a different sized control amplicon, withone-another will indicate the degree of degradation of the sample beingtested. For example, a degraded sample may not contain a largeconcentration of long nucleic acid fragments (e.g., in the 150 or 200base size range), thus decreasing or eliminating the larger controlpeaks (e.g., FIGS. 1B, 1C, 7). In contrast, a nucleic acid moleculesample that is not significantly degraded will generally exhibit threesignals of equal size (e.g., FIG. 1A, FIG. 2A, FIG. 2B, FIG. 2C, FIG.3A, FIG. 3B, FIG. 3C, FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 5A, FIG.5B, FIG. 5C, FIG. 6).

FIG. 8 depicts the results of an assay of a mixed sample containingmultiple mutant BRAF and KRAS nucleic acids in addition to internalcontrols (MCAD). The control peaks are located in the boxes, the BRAFmutant peak (V600E) is located in the oval, and the multiple KRAS peaks(G12R, G12C, G12S, G12A, G12V, G12D, and G13D) are unmarked. In such anassay the individual peaks of each nucleic acid are clearly discernableabove background and from one another. The peak levels also provide aqualitative measure of the amount of mutant nucleic acid in the sample,as described above, with reference to the control peak levels. ExemplaryMCAD primers are set forth in Table 1.

TABLE 1 SEQ ID Primer Nucleotide Sequence NO: MCAD FNED-GTACTTCACAAATTCAAAGACTTATTGTATCC 1 (NED) MCAD F2NED-GTACTTCACAAATTCAAAGACTTATTGTAGCC 2 (NED) MCAD 80RTCAATATTTCTACAGTAATTTTTTTAATTTTTG 3 MCAD R2TCAATATTTCTACAGTAATTTTTTTAATTTTTGTACT 4 80 TG MCAD RCTTGTGTTCTAGTTATTCAATATTTCTACAGTAATTT 5 (100) T MCAD RCTGGAAAAAACGTTAAAGCCCTTTCT 6 (150) MCAD RCTACTATAATAGGCAGTTGCTTAGATTTAATATAAGA 7 (200) GG MCAD RTCTAGGTTAATGTAATTCAAGTAAAGTGGTACTAAA 8 (300) GAAAACAmplification and Detection Methods

Genomic DNA (gDNA), complementary DNA (cDNA), and messenger RNA (mRNA)may be amplified prior to or simultaneous with detection. Illustrativenon-limiting examples of nucleic acid amplification techniques include,but are not limited to, polymerase chain reaction (PCR), reversetranscription polymerase chain reaction (RT-PCR), Transcription-MediatedAmplification (TMA), ligase chain reaction (LCR), strand displacementamplification (SDA), and nucleic acid sequence-based amplification(NASBA). Those of ordinary skill in the art will recognize that certainamplification techniques (e.g., PCR) require that RNA be reversedtranscribed to DNA prior to amplification (e.g., RT-PCR), whereas otheramplification techniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction (U.S. Pat. Nos. 4,683,195, 4,683,202,4,800,159 and 4,965,188 (each of which is incorporated herein byreference)), commonly referred to as PCR, uses multiple cycles ofdenaturation, annealing of primer pairs to opposite strands, and primerextension to exponentially increase copy numbers of a target nucleicacid sequence. In a variation called RT-PCR, reverse transcriptase (RT)is used to make a complementary DNA (cDNA) from mRNA, and the cDNA isthen amplified by PCR to produce multiple copies of DNA. For othervarious permutations of PCR see, e.g., U.S. Pat. Nos. 4,683,195,4,683,202, and 4,800,159; Mullis et al., Meth. Enzymol., 1987, 155: 335;and, Murakawa et al., DNA, 1988, 7: 287 (each of which is incorporatedherein by reference).

Transcription-Mediated Amplification (U.S. Pat. Nos. 5,480,784 and5,399,491, each of which is incorporated herein by reference), commonlyreferred to as TMA, synthesizes multiple copies of a target nucleic acidsequence autocatalytically under conditions of substantially constanttemperature, ionic strength, and pH in which multiple RNA copies of thetarget sequence autocatalytically generate additional copies. See, e.g.,U.S. Pat. Nos. 5,399,491 and 5,824,518 (each of which is incorporatedherein by reference). In a variation described in U.S. Pat. No.7,374,885 (incorporated herein by reference), TMA optionallyincorporates the use of blocking moieties, terminating moieties, andother modifying moieties to aid in the reduction of side-productformation.

The ligase chain reaction (Weiss, R., Science, 1991, 254: 1292,incorporated herein by reference), commonly referred to as LCR, uses twosets of complementary DNA oligonucleotides that hybridize to adjacentregions of the target nucleic acid. The DNA oligonucleotides arecovalently linked by a DNA ligase in repeated cycles of thermaldenaturation, hybridization and ligation to produce a detectabledouble-stranded ligated oligonucleotide product.

Strand displacement amplification (Walker, G. et al., Proc. Natl. Acad.Sci. USA, 1992, 89: 392-396; U.S. Pat. Nos. 5,270,184 and 5,455,166(each of which is incorporated herein by reference)), commonly referredto as SDA, uses cycles of annealing pairs of primer sequences toopposite strands of a target sequence, primer extension in the presenceof a dNTPαS to produce a duplex hemiphosphorothioated primer extensionproduct, endonuclease-mediated nicking of a hemimodified restrictionendonuclease recognition site, and polymerase-mediated primer extensionfrom the 3′ end of the nick to displace an existing strand and produce astrand for the next round of primer annealing, nicking and stranddisplacement, resulting in geometric amplification of product.Thermophilic SDA (tSDA) uses thermophilic endonucleases and polymerasesat higher temperatures in essentially the same method (EP Patent No.0684315, incorporated herein by reference).

Other amplification methods include, for example: nucleic acidsequence-based amplification (U.S. Pat. No. 5,130,238), commonlyreferred to as NASBA; a method that uses an RNA replicase to amplify theprobe molecule itself (Lizardi et al., BioTechnol., 1988, 6: 1197),commonly referred to as Qβ replicase; a transcription-basedamplification method (Kwoh et al., Proc. Natl. Acad. Sci. USA, 1989,86:1173); and a self-sustained sequence replication (Guatelli et al.,Proc. Natl. Acad. Sci. USA, 1990, 87: 1874) (each of which isincorporated herein by reference). For further discussion of certainknown amplification methods see Persing, David H., “In Vitro NucleicAcid Amplification Techniques” in DIAGNOSTIC MEDICAL MICROBIOLOGY:PRINCIPLES AND APPLICATIONS pp. 51-87 (Persing et al., Eds., AmericanSociety for Microbiology, Washington, D.C. (1993)) (incorporated hereinby reference).

Non-amplified or amplified nucleic acids can be detected by anyconventional means. For example, the target nucleic acids and MCADcontrols can be detected by hybridization with a detectably labeledprobe and measurement of the resulting hybrids. Illustrativenon-limiting examples of detection methods are described below.

One illustrative detection method, the Hybridization Protection Assay(HPA) involves hybridizing a chemiluminescent oligonucleotide probe(e.g., an acridinium ester-labeled (AE) probe) to the target sequence,selectively hydrolyzing the chemiluminescent label present onunhybridized probe, and measuring the chemiluminescence produced fromthe remaining probe in a luminometer. See, e.g., U.S. Pat. No.5,283,174; Nelson et al., Nonisotopic Probing, Blotting, and Sequencing,ch. 17 (Larry J. Kricka ed., 2d ed. 1995) (each of which is incorporatedherein by reference).

Another illustrative detection method provides for quantitativeevaluation of the amplification process in real-time. Evaluation of anamplification process in “real-time” involves determining the amount ofamplicon in the reaction mixture either continuously or periodicallyduring the amplification reaction, and using the determined values tocalculate the amount of target sequence initially present in the sample.A variety of methods for determining the amount of initial targetsequence present in a sample based on real-time amplification are wellknown in the art. These include methods disclosed in U.S. Pat. Nos.6,303,305 and 6,541,205 (each of which is incorporated herein byreference). Another method for determining the quantity of targetsequence initially present in a sample, but which is not based on areal-time amplification, is disclosed in U.S. Pat. No. 5,710,029(incorporated herein by reference).

Amplification products may be detected in real-time through the use ofvarious self-hybridizing probes, most of which have a stem-loopstructure. Such self-hybridizing probes are labeled so that they emitdifferently detectable signals, depending on whether the probes are in aself-hybridized state or an altered state through hybridization to atarget sequence. By way of non-limiting example, “molecular torches” area type of self-hybridizing probe that includes distinct regions ofself-complementarity (referred to as “the target binding domain” and“the target closing domain”) which are connected by a joining region(e.g., non-nucleotide linker) and which hybridize to each other underpredetermined hybridization assay conditions. In a preferred embodiment,molecular torches contain single-stranded base regions in the targetbinding domain that are from 1 to about 20 bases in length and areaccessible for hybridization to a target sequence present in anamplification reaction under strand displacement conditions. Understrand displacement conditions, hybridization of the two complementaryregions, which may be fully or partially complementary, of the moleculartorch is favored, except in the presence of the target sequence, whichwill bind to the single-stranded region present in the target bindingdomain and displace all or a portion of the target closing domain. Thetarget binding domain and the target closing domain of a molecular torchinclude a detectable label or a pair of interacting labels (e.g.,luminescent/quencher) positioned so that a different signal is producedwhen the molecular torch is self-hybridized than when the moleculartorch is hybridized to the target sequence, thereby permitting detectionof probe:target duplexes in a test sample in the presence ofunhybridized molecular torches. Molecular torches and a variety of typesof interacting label pairs, including fluorescence resonance energytransfer (FRET) labels, are disclosed in, for example U.S. Pat. Nos.6,534,274 and 5,776,782 (incorporated herein by reference).

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FRET) (see, e.g., U.S. Pat. Nos. 5,631,169& 4,968,103, each of which is incorporated herein by reference). Afluorophore label is selected such that a first donor molecule's emittedfluorescent energy will be absorbed by a fluorescent label on a second,“acceptor” molecule, which in turn is able to fluoresce due to theabsorbed energy.

Alternately, the “donor” protein molecule may simply utilize the naturalfluorescent energy of tryptophan residues. Labels are chosen that emitdifferent wavelengths of light, such that the “acceptor” molecule labelmay be differentiated from that of the “donor.” Since the efficiency ofenergy transfer between the labels is related to the distance separatingthe molecules, the spatial relationship between the molecules can beassessed. In a situation in which binding occurs between the molecules,the fluorescent emission of the “acceptor” molecule label should bemaximal. A FRET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorometer).

Another example of a detection probe having self-complementarity is a“molecular beacon.” Molecular beacons include nucleic acid moleculeshaving a target complementary sequence, an affinity pair (or nucleicacid arms) holding the probe in a closed conformation in the absence ofa target sequence present in an amplification reaction, and a label pairthat interacts when the probe is in a closed conformation. Hybridizationof the target sequence and the target complementary sequence separatesthe members of the affinity pair, thereby shifting the probe to an openconformation. The shift to the open conformation is detectable due toreduced interaction of the label pair, which may be, for example, afluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beaconsare disclosed, for example, in U.S. Pat. Nos. 5,925,517 and 6,150,097(each of which is incorporated herein by reference).

Other self-hybridizing probes are well known to those of ordinary skillin the art. By way of non-limiting example, probe binding pairs havinginteracting labels, such as those disclosed in U.S. Pat. No. 5,928,862(incorporated herein by reference) can be adapted for use in the methodsof the present invention. Probe systems used to detect single nucleotidepolymorphisms (SNPs) can also be utilized in the present invention.Additional detection systems include “molecular switches,” as disclosed,for example, in U.S. Publ. No. 20050042638 (incorporated herein byreference). Other probes, such as those comprising intercalating dyesand/or fluorochromes, are also useful for detection of amplificationproducts methods of embodiments of the present disclosure. See, e.g.,U.S. Pat. No. 5,814,447 (incorporated herein by reference).

Any of these compositions, alone or in combination with othercompositions of the present disclosure, may be provided in the form of akit.

While the present invention has been described and shown in considerabledetail with reference to certain illustrative embodiments, includingvarious combinations and sub-combinations of features, those skilled inthe art will readily appreciate other embodiments and variations andmodifications thereof as encompassed within the scope of the presentinvention. Moreover, the descriptions of such embodiments, combinations,and sub-combinations is not intended to convey that the inventionsrequires features or combinations of features other than those expresslyrecited in the claims. Accordingly, the present invention is deemed toinclude all modifications and variations encompassed within the spiritand scope of the following appended claims.

EXAMPLE

A multiplex assay was prepared to determine whether a patient has a KRASor BRAF mutation using DNA extracted from an FFPE tissue. The exemplaryassay tests for 6 mutations located on codon 12 and 1 mutation on codon13 of KRAS, and mutations on codon 600 of BRAF. The target mutationsmutations differ by 1 base pair (missense mutation) causing one aminoacid to be replaced with another, for example, KRAS G12R, KRAS G12S,KRAS G12C, KRAS G12D, KRAS G12A, KRAS G12V, KRAS G13D, and BRAF V600E.

Microtomy was used to slice the sample into fine sections. DNA wasextracted using the QIAMP FFPE DNA Extraction Kit (Qiagen GmbH, Hilden,Germany). The sample was contacted with the KRAS/BRAF PCR Mix which alsocontained an MCAD control amplification reagent mix as an internalcontrol. 2.5 uL of DNA was amplified using ARMS PCR (12.5 uL TotalVolume), purified in a PERFORMA® spin column and then 3 uL of purifiedamplicon was run on a genetic analyzer (3130XL Genetic Analyzer, LifeTechnologies Corp., Carlsbad, Calif.) using 15 uL ABI Hi-Di and a LIZ600Size Standard (18 uL Total Volume).

MCAD Primers amplified DNA to create 3 peaks at 100, 150 and 200 bases(FIG. 8). KRAS and BRAF Mutant Peaks are located between the MCAD 100and 150 base peaks (FIG. 8).

The assay was balanced using gDNA from cell lines containing mutantheterozygous or homozygous DNA mixed with wildtype gDNA (FIG. 4A, FIG.4B, FIG. 4C, FIG. 4D, FIG. 5A, FIG. 5B, FIG. 5C). 20% mixture of mutantto wild-type gDNA was used to balance with the MCAD peaks (FIG. 5A, FIG.5B, FIG. 5C).

What is claimed is:
 1. A kit for evaluating a biological samplecontaining potentially degraded DNA, comprising a control amplificationreagent and a target amplification reagent, wherein the controlamplification reagent comprises two or more pairs of amplificationoligonucleotides capable of amplifying a medium chain acyl-coenzyme Adehydrogenase (MCAD) nucleic acid molecule or a complement thereof, andwherein the target amplification reagent comprises one or more pairs ofamplification oligonucleotides capable of amplifying a non-MCAD targetgene of interest or a complement thereof, wherein each pair of controlamplification reagent amplification oligonucleotides is capable ofamplifying a region of the MCAD nucleic acid molecule to produce an MCADamplicon, and wherein each MCAD amplicon produced by each pair ofamplification oligonucleotides is detectably distinguishable from theMCAD amplicon produced by each other pair of amplificationoligonucleotides on the length of each MCAD amplicon, and wherein one ormore of the amplification oligonucleotides is/are labeled.
 2. The kit ofclaim 1, comprising three or more pairs of amplificationoligonucleotides capable of amplifying the MCAD nucleic acid molecule ora complement thereof.
 3. The kit of claim 1, wherein each of the two ormore pairs of control amplification reagent amplificationoligonucleotides comprises a sense amplification oligonucleotide and anantisense amplification oligonucleotide, and wherein the senseamplification oligonucleotide of each of the two or more pairs ofcontrol amplification reagent amplification oligonucleotides comprisesthe same oligonucleotide sequence.
 4. The kit of claim 1, wherein eachof the two or more pairs of control amplification reagent amplificationoligonucleotides comprises a sense amplification oligonucleotide and anantisense amplification oligonucleotide, and wherein the antisenseamplification oligonucleotide of each of the two or more pairs ofcontrol amplification reagent amplification oligonucleotides comprisesthe same oligonucleotide sequence.
 5. The kit of claim 1, wherein thebiological sample comprises a tissue sample.
 6. The kit of claim 5,wherein the tissue sample comprises a formalin-fixed paraffin-embedded(FFPE) tissue sample.
 7. The kit of claim 1, wherein the targetamplification reagent comprises a reagent specific for wild-type ormutant v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), V-Ki-ras2Kirsten rat sarcoma viral oncogene homolog (KRAS), epidermal growthfactor receptor (EGFR), phosphatidylinositol 3-kinase (PIK3CA),phosphatase and tensin homolog (PTEN), v-akt murine thymoma viraloncogene homolog (AKT), anaplastic lymphoma kinase (ALK), mast/stem cellgrowth factor receptor (c-Kit), neuroblastoma RAS viral oncogene homolog(NRAS), met proto-oncogene hepatocyte growth factor receptor (c-Met),prostate cancer gene 3 (PCA3), prostate specific membrane antigen(PSMA), prostate specific antigen (PSA), tumor protein 53 (TP53),Echinoderm microtubule-associated protein-like 4 (EML4), EML4-ALKfusions, androgen regulated gene-ETS family member gene fusions, RAFgene fusions, breakpoint cluster region −V-abl Abelson murine leukemiaviral oncogene homolog 1 fusions (BCR-Abl), cytochrome P450 2D6(CYP2D6), cytochrome P450 2C19 (CYP2C19), cytochrome P450 2C9 (CYP2C9),vitamin K epoxide reductase complex subunit 1 (VKORC1), thiopurinemethyltransferase (TMPT), bilirubin UDP-glucuronosyltransferase isozyme1 (UGT1A1), and/or ATP-binding cassette sub-family B member 1 (ABCB1),or combinations thereof.